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Colloidally Stabilized Magnetic Carbon Nanotubes Providing MRI Contrast in Mouse Liver Tumors Yue Liu, Benjamin W. Muir, Lynne J Waddington, Tracey M. Hinton, Bradford A. Moffat, Xiaojuan Hao, Jieshan Qiu, and Timothy C Hughes Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501706x • Publication Date (Web): 04 Feb 2015 Downloaded from http://pubs.acs.org on February 15, 2015

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Colloidally Stabilized Magnetic Carbon Nanotubes Providing MRI Contrast in Mouse Liver Tumors Yue Liu, a,b Benjamin W. Muir,b Lynne J. Waddington,c Tracey M. Hinton,d Bradford A. Moffat,e Xiaojuan Hao,*b Jieshan Qiu,*a and Timothy C. Hughes*b a

Carbon Research Laboratory, Liaoning Key Lab for Energy Materials and Chemical

Engineering, State Key Lab of Fine Chemicals, Dalian University of Technology, 116000, China. b

CSIRO Manufacturing Flagship, Clayton 3168, Australia.

c

CSIRO Manufacturing Flagship, Parkville 3052, Australia

d

CSIRO Biosecurity Flagship, Australian Animal Health Laboratory, Geelong 3220, Australia

e

The University of Melbourne, Department of Radiology, Parkville, Victoria 3050, Australia.

ABSTRACT. The use of medical imaging contrast agents may lead to improved patient prognosis by potentially enabling an earlier detection of diseases and therefore an earlier initiation of treatments. In this study, we fabricated superparamagnetic iron oxide (SPIO)

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nanoparticles within the inner cavity of multiwalled carbon nanotubes (MWCNTs) for the first time; thereby ensuring high mechanical stability of the nanoparticles. A simple, but effective, self assembled coating with RAFT diblock copolymers ensured the SPIO-MWCNTs have a high dispersion stability under physiological conditions. In vivo acute tolerance testing in mice showed a high tolerance dose up to 100 mg kg-1. Most importantly, after administration of the material a 55% increase in tumor to liver contrast ratio was observed with in vivo MRI measurements compared to the pre-injection image enhancing the detection of the tumor.

KEYWORDS. MWCNTs, MRI contrast agent, RAFT polymerization, microwave reaction, superparamagnetic iron oxide

Introduction Carbon nanotube (CNT) based nanotechnologies have been rapidly developing for a variety of applications since their discovery by Iijima in 19911. The combination of CNTs with nanomagnets exhibits unique properties leading to a potential for applications in cancer diagnosis and therapy. The most promising applications of these hybrid nanomaterials include targeted or controllable drug delivery systems2, magnetic particle/fluid hyperthermia anti-cancer therapy3, and magnetic resonance imaging (MRI) agents4. To date, there are limited reports on the use of magnetic CNTs as MRI contrast agents4-10. In 2005, the nanoscale loading and confinement of Gd3+ ion clusters within ultra-short single-walled carbon nanotubes (SWCNTs) was reported as a potential T1-weighted contrast agent7. Afterwards, SWCNTs with iron catalyst impurities inside the tubes were investigated for use as T2-weighted contrast agents6. Due to the toxicity of Gd(III)11, it has to be strongly complexed in a suitable chemical ligand when used clinically. As


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an alternative paramagnetic material, superparamagnetic iron oxide (SPIO) nanoparticles and ultra-small SPIO (USPIO) nanoparticles that are considered to be relatively well tolerated to the body have been explored as T2-weighted contrast agents8. CNT-SPIO nanostructures have also been investigated with comparatively high T2 relaxivities and low cytotoxicity4. Functionalized CNTs containing potential targeting ligands such as folic acid and galactose have shown to be effective contrast agents for cancer cell specific imaging9, 10. Currently there is a concern about the cytotoxicity of CNTs12 partly due to their tendency to form aggregates, which may impede their uptake, delivery, and clearance from the body. As such, CNTs need to be modified before use for biomedical applications to inhibit aggregation and improve their vascular biocompatibility. Poly(ethylene glycol) (PEG) is one of the most effective polymers utilized to modify nanomaterials with highly hydrophilic and low protein fouling properties, which can result in increased blood circulation half-lives13. CNT based drug carriers composed of branched PEG chains and chemotherapeutic drugs used as ‘longboat’ delivery system have shown improved efficiency to target tumor cells14. CNTs grafted with PEG chains have a blood half-life of 12-13 hours which appears to be optimal to balance tumor-tonormal organ uptake ratios in photothermal therapy applications15. However, no detailed in vivo studies of immune or inflammatory response have been reported in animals with PEG-modified CNTs, though there was a report that delivery of PEGylated cationic, shell-crosslinked-needlelike nanoparticles to mouse airway showed significantly less inflammation which was PEG dosedependent 16. Among the investigations of CNT-Fe3O4 hybrid materials as imaging contrast agents, magnetic components are usually decorated outside of the CNTs, and as such the effective components of the contrast agents (i.e. magnetic materials) could potentially detach during their


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use. It has been reported that metal salts, palladium, platinum, silver and gold nanoparticles grown on CNTs by thermal decomposition were mostly detached from the CNTs17, indicating poor adhesion of the nanoparticles to the CNTs. Sitharaman et al. have reported the use of Gd3+ ion clusters within ultra-short single-walled carbon nanotubes as nanocapsules for MRI contrast agents7. There have been earlier attempts to incorporate magnetic particles into CNTs18, but the size of the nanoparticles make them unsuitable for NMR imaging. Thus, to the best of our knowledge, there is no report about the preparation of SPIO nanoparticles encapsulated inside CNTs for MRI contrast agents. With this in mind, the aim of this study was to synthesize and characterize CNTs containing SPIO nanoparticles (CNT-Fe3O4). It was hypothesized that Fe3O4 nanoparticles synthesized in situ by a microwave-assisted reduction method would introduce SPIO nanoparticles predominantly inside the CNTs. In addition, the CNT-Fe3O4 hybrid materials when coated with diblock copolymers, prepared by the RAFT polymerization, would improve their aqueous and buffer (PBS) dispersion stability and thus their circulation time in vivo. The MWCNTs hybrids were characterized by TGA, Zeta-potential, TEM, and XRD, while the magnetic properties of the products were investigated via relaxivity measurements in a 3T MRI scanner and superconducting quantum interference device (SQUID) measurements. An Alamar Blue cell viability assay was used to investigate the cytotoxicity of the magnetic MWCNTs hybrids. MRI measurements in a diseased mouse model were carried out to assess their performance in a clinically relevant model imaging tumor lesions in vivo. Experimental Section Materials. Multiwalled carbon nanotubes (MWCNTs) were purchased from Chengdu Organic Chemicals Co., Ltd, China, with an average outer diameter of 30-60 nm, inner diameter of 20-50


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nm, and a length of 1-10 µm. Poly(ethylene glycol) methacrylate (PEGMA; MW 526) was purchased from Aldrich and the inhibitor was removed by passing it through a column packed with quaternary ammonium anion-exchange resin (Aldrich). RAFT agent 4-cyano-4(thiobenzoylthio) pentanoic acid (CTA; 97%) was purchased from Strem Chemicals and used as received. 2-(Methacryloxy) ethyl trimethyl ammonium chloride (METAC; 75%) was purchased from Aldrich and purified by precipitating into acetone before use. 2, 2´-Azobis-(2methylpropionitrile) (AIBN) was purchased from Aldrich. Nitric acid (70%) and sulfuric acid (95-98%) from Chem-Supply, citric acid, acetone, methanol, and diethyl ether from Merck, hydrazine hydrate (N2H4·H2O) from Sigma-Aldrich, ferric acetyl acetonate (Fe(acac)3) from Aladdin were of analytical reagent grade and used without further purification. Acid treatment of MWCNTs. MWCNTs (10 g) were chemically functionalized in a mixture of water (60 mL), sulfuric acid (95-98%, 180 mL), and nitric acid (70%, 60 mL) (1: 3: 1) for 4 h at 100 oC and then rinsed with Milli-Q water via filtration until the filtrate was neutral. The collected MWCNTs (designated as o-CNTs) were dried in the vacuum oven at 60 oC. All oCNTs prepared were further treated by citric acid (28 g) in 300 mL Milli-Q water for 2 h at 80 oC to generate more -COOH groups (designated as c-CNTs). After reaction, c-CNTs were rinsed with Milli-Q water via filtration until the filtrate was neutral and then dried. Preparation of magnetic MWCNTs. To prepare magnetic MWCNTs, Fe(acac)3 (8 g) was firstly dissolved in EtOH (300 mL). After removing the precipitates by filtration, the Fe(acac)3 solution was mixed with c-CNTs (8 g). Ultrasonication and stirring were alternately applied to mix them for 30 min. The mixture was then dried by Rotavapor to remove EtOH to get the solid powder (designated as CNT-Fe(acac)3).


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A microwave reactor was used to reduce Fe(acac)3 to Fe3O4. CNT-Fe(acac)3 (0.4 g) and N2H4 (1 mL) were dispersed in H2O (16 mL) in a microwave reactor vial under ultrasonication for 10 min. After pre-stirring for 2 min, the reaction was carried out in microwave reactor at 100 oC for 20 min. The product was washed by Milli-Q water via filtration until the filtrate was neutral and dried overnight in vacuum oven (designated as CNT-Fe3O4). Preparation of dispersible magnetic MWCNTs. Diblock copolymers (PMETAC-bPEGMA) with different PEGMA degree of polymerization (DP) were prepared as previously reported19. CNT-Fe3O4 (100 mg) was initially suspended in Milli-Q water (50 mL) by ultrasonication for 30 min in the presence of diblock copolymer (100 mg), followed by rinsing with Milli-Q water (50 mL, 3 times) via filtration to remove unattached polymer. The generated product was designated as CNT-Fe3O4-polymer. Characterization. Nuclear magnetic resonance (NMR) was employed to determine monomer conversion and DP of block polymers using a Bruker Av 400 NMR spectrometer. 1H NMR spectra were recorded in deuterium methanol (MeOD). XRD was performed on the SPIOs for phase determination using a PANalytical X’Pert powder diffractometer fitted with a Cu target tube operated at 40 kV and 40 mA. Thermal stability of the hybrid CNT samples was assessed using a thermogravimetry analysis (TGA) instrument (Mettler-Toledo TGA/SDT851). Experiments were conducted on 5-10 mg of samples heated in a flowing air at a heating rate of 10 °C min-1 from 30 to 800 °C. Dispersion tests were carried out as below: 4 mg sample was dispersed in 1 mL Milli-Q water or PBS solution in a glass vial. Ultrasonication was used to ensure a good dispersion. Subsequently, the glass vial was put on a plain board for photographic imaging.


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Zeta-potential measurements were performed at 25 °C using a Zetasizer-Nano instrument (Malvern, UK). TEM was carried out using Tecnai12 transmission electron microscopy (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. Magnetic property measurements were carried out on a Quantum Design MPMS 5 SQUID magnetometer10. Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, Varian-Vista instrument) was used to determine the iron content of samples. MRI relaxivity measurement. A high-throughput MRI screening technique was used to evaluate the NMR relaxation properties of the sample solutions at 3T using a method similar to that previously reported 20. Briefly, for MRI relaxivity measurements, 1 mL of each CNT-Fe3O4polymer solution was placed in a 96 well plate and serially diluted to measure T2 and calculate relaxivities. For quantifying the spin-spin relaxation rates (R2 = 1/T2) a multiple spin echo sequence was used to acquire 32 images at echo times (TE) ranging from 11.5 to 310.5 ms with a repetition time (TR) of 3 s Toxicity testing. To investigate the cytotoxicity of the CNT-Fe3O4-polymer product, an Alamar Blue cell viability assay was performed19. Briefly, Huh7 cells (kindly supplied by VIDRL, Australia) were grown in Dulbecco's modified eagle medium (DMEM) and CHO-GFP cells ((kindly received from K. Wark; CSIRO CMHT Australia) were grown in MEMα modification supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, 0.01% penicillin and 0.01% streptomycin at 37 oC with 5% CO2 and subcultured twice weekly. Both cell lines were seeded at 1×104 cells in 96-well tissue culture plates in triplicate and grown overnight at 37


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ºC with 5% CO2. The MWCNTs samples were added to cells and incubated for 72 h before toxicity measurement. Results are presented as a percentage of untreated cells. All animal procedures were conducted in compliance with all the laws, regulations, and guidelines of the National Institutes of Health (NIH) and with the approval of Molecular Imaging, Inc.’s Animal Care and Use Committee. Molecular Imaging, Inc. is an AAALAC accredited facility. 5-6 week old Harlan BALB/c mice (BALB/cAnNHsd) were used for acute maximum tolerated dose studies and allowed to acclimatize for 5 days before being used. Three mice were dosed individually by body weight on the day of treatment at 100 mg kg-1. Animals were housed for 5 days after a single dose to measure any delayed toxicity response. Upon euthanasia, all mice were necropsied to provide a general assessment of target organs for toxicity. MRI imaging in vivo. 7-8 week old Female Harlan BALB/c mice (BALB/cAnNHsd) weighing ≥14.7 g were used for this study. The murine carcinoma inoculations and MRI measurements were carried out following the procedure as previously reported10. Briefly, the surgical area was disinfected and a 1 cm incision was made in the skin directly above the spleen. A ≤ 2 mm incision was made in the peritoneum so that the caudal end of the spleen could be exposed. 10 µL of a 30% C26 brei was injected into the spleen. After at least 1 minute, the spleen was removed following arterial ligation and the peritoneum was closed using absorbable suture. The animals were then allowed to recover on a re-circulating water heating pad. Once fully recovered, the mice were returned to their respective cages. Animals were sorted into groups of three and imaged using standard fast spin-echo multislice sequence (fsems) anatomical images to measure total liver metastasis tumour burden. After standard MRI preparation (optimization of shimming, pulse power calibration, line width


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determination, and scout images to locate the tumor), a T2-weighted (T2w) FSEMS was used to image the tumor volumes. The acquisition parameters were: repetition time = 3 s, 8 echoes, echospacing = 14.2 ms, k-space centred on the fourth echo, and 26 averages. A 30 mm square field of view was used with an image matrix of 128 × 128 and 12 contiguous, 1.5 mm-thick transaxial slices that covered the entire tumor. Contrast imaging and nanoparticle administration occurred on day 10 post implantation of tumor. Two MRI scans were acquired for each animal: one pre-treatment scan, after which the mice were intravenously injected as indicated in the protocol. The mice were then imaged again immediately after injection to obtain a post-treatment scan. After standard MRI preparation (optimization of shimming, pulse power calibration, line width determination, and scout images to locate the liver), a T1-weighted (T1w) spin-echo multislice sequence (SEMS) was used to image the area of interest (from the bottom of the lungs through to the bottom of the liver). The acquisition parameters were: recycle time = 500 ms, echo time = 9.5 ms, inversion time = 100 ms, and 4 averages. A 30 mm square field of view was used with an image matrix of 128 × 128 and 6 contiguous, 1.5 mm-thick transaxial slices that covered the entire liver volume. All images were reconstructed using custom written scripts in Matlab and the tumor volumes and signal intensities were defined using Amira image processing package. Results and discussion Synthesis of CNT-Fe3O4-polymer composite. The combination of SPIO and MWCNTs has attracted extensive interest from different fields21-23. However, in some cases, the attached nanoparticles outside of MWCNTs were unstable17. Therefore, encapsulation of the nanoparticles inside the MWCNTs is preferred. In the present work, we have synthesized


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polymer-modified MWCNTs composite with SPIO nanoparticles encapsulated inside the central cavity of the CNTs (Scheme 1).

Scheme 1. CNT-Fe3O4-polymer preparation The synthetic route from an iron precursor to iron oxide nanoparticles via a microwave reaction has been previously explored24. Moreover, microwave reactions have previously been used in the reduction reaction to convert iron (III) acetylacetonate to Fe3O4 nanoparticles outside of CNTs25. In our case, MWCNTs with a large inner diameter were used to fabricate magnetic composites using Fe(acac)3 as the iron precursor. In addition, as the outside walls of MWCNTs have been pre-treated by mixed acids to produce carboxyl groups (hydrophilic surface), the SPIO nanoparticles tended to attach to the more hydrophobic central cavity of the MWCNTs due to hydrophobic interactions26. XRD analysis was used to examine the crystal phase of nanoparticles as shown in Figure 1a. All the measured diffraction peaks matched the standard cubic phase of Fe3O4 (JCPDS Card No. 19-0629) and graphite (JCPDS Card No. 76-1651). The average crystallite size of Fe3O4 nanoparticles is calculated to be about 16 nm by the Scherer equation27.


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Figure 1. XRD pattern of CNT-Fe3O4 (a), TGA curves of CNT-Fe3O4-polymer (b), and the mass loss vs PEGMA DP plot (c)

In order to improve the dispersion stability of magnetic MWCNTs in saline, diblock copolymers were employed to modify the surface of MWCNTs (Scheme 1). The synthesis of the diblock polymers and their coating onto CNTs are described in our previous publication19. The cationic PMETAC block was designed to electrostatically bind to the negatively charged oxidized CNTs while the second functional PEGMA block was designed to improve the aqueous dispersion stability of the hybrid material. Diblock copolymers were prepared with different PEGMA block lengths (DP = 20, 60, 89, and 118, named P1 to P4, respectively) and the same PMETAC block length (DP 10)19. TGA was used to verify the existence of a diblock copolymer layer on the surface of CNT-Fe3O4. As shown in Figure 1b, TGA analysis of the diblock polymer shows that the majority of degradation occurs between 220 to 500 oC. The CNT-Fe3O4-polymer samples also showed a significant mass loss over the same range (mass loss range of 16.6 to 34.5%). In contrast, the unmodified MWCNTs had essentially no mass loss over this temperature range. The CNT-Fe3O4-polymer samples showed a linear increase in mass loss with increasing DP of PEGMA (Figure 1c) consistent with our earlier report19 on the CNT-polymer samples.


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Also, the mass loss of CNT-Fe3O4-polymer samples was similar to that of corresponding CNTpolymer samples (mass loss range of 10.3 to 34.9%) which indicated that the presence of Fe3O4 did not significantly affect the binding between MWCNTs and diblock copolymers. Zeta-potential is an efficient and direct way to monitor the change in the surface charge of materials. As shown in Figure 2a, acid-treated MWCNTs have a greater negative charge than the pristine MWCNTs. Due to the positive charge contribution of Fe(acac)3, the composite CNTFe(acac)3 changed from negative to positive charge. After being reduced to Fe3O4, the composite displayed a negative charge again. The increase in Zeta-potential of the CNT-Fe3O4-polymer composites was attributed to the effect of the block polymer.

Figure 2. Zeta-potential (a) and TEM images of c-CNTs (b), CNT-Fe3O4-P4 (c), and negatively stained CNT-Fe3O4-P4 (d, e)


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TEM is commonly used to examine the structures of various nanomaterials. The images of cCNTs and the CNT-Fe3O4-P4 were shown in Figures 2b and 2c, respectively. Compared with cCNTs, Fe3O4 particles were clearly observed inside the MWCNTs. It is proposed that the absence of nanoparticles outside of MWCNTs was due to the lack of binding force between the particles and the outer surface of the MWCNTs (both have a negative Z-potential). The thin layer of polymer outside the MWCNTs was not observed at a low TEM resolution. However, upon negatively staining with potassium phosphotungstate a polymer layer could be clearly observed due to its significantly different electron density from the wall of MWCNTs as shown in Figure 2d. Comparing Figures 2c and 2d, the particles were clearly visible without staining but the polymer layer was difficult to see, which could be clearly seen after negatively staining. However, the high electron density of the negative staining may obscure the nanoparticles. Further evidence that the objects inside the CNTs central cavity are nanoparticle clusters can be obtained by comparing the TEM image 2d to those of the negatively stained CNT-polymer composites previously reported19. TEM image with several full length nanotubes of CNT-Fe3O4P4 sample was presented in Figure 2e, showing that the average length of the nanotubes was about 400 nm. A critical requirement for contrast agents is their stability in aqueous media, especially in buffers such as PBS to ensure that they can be successfully delivered via an intravenous bolus to the targeted organ in the body. Dispersion stability tests of the composites in water and PBS buffer solution were carried out to evaluate the effect of the diblock polymer coatings. The evaluation was conducted by the visual observations over 7 days and recorded photographically with the end point images shown in Table 1.


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Table 1. Stability of various MWCNTs composites in water and PBS In PBS

In water

CNT-

CNT-

CNT-

CNT-

CNT-

CNT-

CNT-

Fe3O4

Fe3O4-P1a)

Fe3O4-P2

Fe3O4-P3

Fe3O4-P4

Fe3O4

Fe3O4-P4

10 min

30 min

3 hours

7 daysb)

7 days

7 days

7 days

a)

P1-P4 are block polymers consisting of PMETAC block (DP = 10) and PEGMA block with varying DP (20, 60, 89, 118, respectively); b) 7 days was the maximum testing time.

It can be seen from Table 1 that both uncoated CNT-Fe3O4 and CNT-Fe3O4-P4 maintained stable dispersions in water for more than 7 days. However, in PBS, the CNT-Fe3O4 suspension precipitated quickly in less than 10 min, indicating a very poor dispersion stability in buffer and suggesting that uncoated CNT-Fe3O4 is not suitable to be used in body. In contrast, polymer coated samples (CNT-Fe3O4-polymer) displayed better dispersion stability due to the presence of the polymer layer. The hydrophilicity of the copolymer coating greatly affects the stability of CNT-Fe3O4-polymer samples particularly in PBS solution. Among a series of block polymers synthesized, the polymers with a relatively low DP showed inefficient hydrophilicity, resulting in a poor dispersion stability, as observed in the case of CNT-Fe3O4-P1-3 composites. However, there is a clear trend that the duration that the materials maintained a stable dispersion in PBS solution significantly increased with increasing DP of PEG block, which indicated that the longer


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polymer blocks showed a significant improvement in dispersion stability in PBS buffer. CNTFe3O4-P4 exhibited a long term dispersion stability in aqueous PBS solution for at least 7 days. These results are consistent with previously reported dispersion tests of composites without Fe3O4 nanoparticles19, indicating that the presence of Fe3O4 nanoparticles inside the central cavity of CNTs has little effect on their dispersion stability. Magnetic properties of CNT-Fe3O4-polymer. The CNT-Fe3O4-P4 composite with high water and PBS dispersion stability was selected for further characterization. A SQUID magnetometer was used to measure the magnetic properties of the CNT-Fe3O4-P4 that originated from Fe3O4 nanoparticles inside the MWCNTs. The hysteresis loop of the CNT-Fe3O4-P4 composite is shown in Figure 3a. It is obvious that the sample has a near-saturation magnetization value, i.e. ~11 emu g-1 above 20 000 Oe. Furthermore, the superparamagnetic nature of the hybrid material was confirmed by the narrow magnetization hysteresis loop, which is typical for SPIO nanoparticles. Figure 3b shows the zero-field-cooled and field-cooled (ZFC/FC) curves of CNT-Fe3O4-P4 measured between 10 and 350 K at an applied field of 100 Oe. As the temperature increases from 10 to 350 K, the ZFC magnetization increases first and then decreases after reaching maximum around 200 K, which corresponds to the blocking temperature (TB). This result further confirms that the CNT-Fe3O4-P4 has a superparamagnetic behaviour at room temperature. In comparison, the FC magnetization decreases as the temperature increases. It is envisaged that the difference between the ZFC magnetization and the FC magnetization below TB is caused by energy barriers of the magnetic anisotropy28, which

makes the CNT-Fe3O4-P4 material possess magnetic

properties, suggesting that it would be a suitable agent to influence the relaxivity of water in MRI experiments.


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Figure 3. Hysteresis loops (a) and ZFC/FC curves (b) of CNT-Fe3O4-P4; (c) MRI relaxation results of CNT-Fe3O4-P4; (d) Estimated R2 of CNT-Fe3O4-P1-3 based on TGA analysis and measured R2 of CNT-Fe3O4-P4

The R2 relaxivity (mM−1s−1) is a measurement of the effectiveness of an MRI contrast agent in reducing the T2 relaxation time, typically in an aqueous environment. After being dispersed in water and fixed within agarose gel, the samples at different concentrations were measured in multi-well plates inside a MRI scanner. The corresponding R2 relaxivities were calculated by plotting the signal response as a function of iron concentration. As shown in Figure 3c, the obtained relaxation rate, R2 (1/T2) changed linearly with increasing Fe concentration of the


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nanocomposites. The gradient of this plot shows that the CNT-Fe3O4-P4 composite has generated a MRI contrast on T2-weighted spin-echo sequences with R2 of 85 mM-1s-1. The modification of CNT-Fe3O4 with various polymers of different DP was studied to examine the effect of polymer chain length on the efficiency as contrast agent. The iron concentration of CNT-Fe3O4-P4 was measured directly by ICP-OES, while the iron concentrations of the other samples CNT-Fe3O4-P1-3 were calculated according to the TGA results and the ICP-OES result of CNT-Fe3O4-P4 based on the ratio of polymer mass loss of CNT-Fe3O4-P1-3 to CNT-Fe3O4-P4. As shown in Figure 3d, uncoated CNT-Fe3O4 exhibited a higher R2 relaxivity than the CNTFe3O4-polymer samples. However, its poor dispersion stability in saline makes it unsuitable as an MRI contrast agent. With the increasing DP of polymer, the R2 relaxivity decreased, indicating that the polymer coating may inhibit the interaction between Fe3O4 nanoparticles and water. However, CNT-Fe3O4-P4 showed an acceptable R2 relaxivity, thus can be used as a suitable contrast agent due to its improved dispersion stability in saline. Toxicity assessment of CNT-Fe3O4-Polymer hybrid. There is a concern about the potential toxicity of MWCNTs due to their nano structure and hydrophobic properties29. In addition to improving aqueous dispersion stability, one of the aims of surface functionalization is to reduce the cytotoxicity of MWCNTs. As shown in Figure 4a and 4b, after exposure to the CNT hybrid materials, there was no significant decrease in cell viability for both kinds of cell lines up to a concentration of 200 µg mL-1, showing that CNT-Fe3O4 modified by diblock copolymers displayed a low in vitro cytotoxicity. The in vitro tests have demonstrated that the hybrid materials possessed a low cytotoxicity to CHO-GFP and Huh7 cells, therefore, acute maximum tolerated dose determination was


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performed on live animals. Mice were injected with CNT-Fe3O4-P4 at 100 mg kg-1 and monitored for acute toxicity. The mice were monitored for 5 days after a single dose to assess them for delayed toxicity response shown in Table 2. The animals remained healthy, showing no weight loss, and behaved normally over the study period. After 5 days, the animals were euthanized, and necropsies were performed with no remarkable findings observed, indicating that CNT-Fe3O4-P4 possessed low toxicity in a live rodent model.

Figure 4. Cytotoxicity results of MWCNT composites with CHO-GFP (a) and Huh7 (b) cell lines; In vivo MRI images of mouse liver pre- and post-injection of CNT-Fe3O4-P4 at a dose of 100 mg kg-1 (white arrows indicate tumors) compared to internal standard (water, top right)


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Table 2. In vivo tolerance performance of CNT-Fe3O4-P4 Time

Animal

Observation

0 min

1, 2, 3

Mice appear normal.

1 day

1, 2, 3

Mice appear normal.

2 days

1, 2, 3

Mice appear normal.

3 days

1, 2, 3

Mice appear normal.

4 days

1, 2, 3

Mice appear normal.

1, 2, 3

Animals euthanized. Necropsies performed, no remarkable findings, lungs pink in color.

5 days

In vivo MRI imaging with CNT-Fe3O4-P4. The detailed characterization of this new nanomaterial has shown that the CNT-Fe3O4-P4 is an excellent MRI contrast agent candidate with good relaxivity and low toxicity. Further evaluation of contrast efficiency of CNT-Fe3O4-P4 was performed in a liver-tumor mice model. A 10 mg mL-1 suspension was administrated to mice via a tail vein injection at a dose of 100 mg per kg body weight. The contrast levels of liver regions of interest (ROIs) and tumor ROIs were compared to the internal standard (water). The contrast ratios were summarized for each animal and the signal ratio (tumor signal/liver signal) of pre- and post- contrast agent injection was calculated, with the change (pre to post) used as the end point for evaluation of the contrast enhancement. Figure 4c and 4d show the T2-weighted MRI images of a cancerous mouse liver before and after administration of CNT-Fe3O4-P4. SPIOs are known to act as T2 contrast agents and darken T2 weighted MR images30-32, hence after injection, the background became much darker, making tumors clearly visible. As shown in Table 3, the data demonstrated a meaningful increase in


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tumor/liver contrast ratio after administration of CNT-Fe3O4-P4, with no increase in contrast observed for the standard positive control, Magnevist. The contrast enhancement was mainly due to a decrease in the normal liver signal, relative to minimal change in tumor signal. This is consistent with a readily uptake of CNT-Fe3O4-P4 in the healthy liver tissue, compared with minimal uptake in diseased liver tumors. The sample showed a contrast enhancement with 55% increase in the tumor/liver contrast ratio compared to pre-injection. In comparison, the standard control, Magnevist showed signal modulation in both liver and tumor, resulting in a slight decrease in tumor/liver contrast ratio after administration. Overall, the MRI results indicate that the CNT-Fe3O4-P4 can be used to image the internal structures of the body and the contrast between liver tumours and healthy tissue can be greatly improved.

Table 3. Summary of average contrast signals for liver and tumor and tumor/liver contrast ratio compared to water (internal standard) Liver

Tumor

Tumor/Liver

Pre/ Post

Pre/ Post

Pre/ Post

137/ 83

374/ 347

2.7/ 4.2

5988/ 15169

11107/ 21404

1.9/ 1.4

Dose (concentration) CNT-Fe3O4-P4 0.18 mmol (Fe) kg-1 Magnevist 0.4 mmol (Gd-DTPA) kg-1

Conclusions A series of CNT-Fe3O4-polymer hybrid materials with improved dispersion stability in PBS were synthesized for potential use as MRI contrast agents. Successful completion of the reaction steps was monitored and confirmed by the TGA and Zeta-potential analyses. In order to improve


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the stability of the SPIO nanoparticles in the composites, the SPIO nanoparticles were encapsulated inside the central cavity of MWCNTs via an in situ reduction. The XRD analysis has confirmed that the encapsulated particles are Fe3O4. The self-assembled coating formed from the diblock copolymers, prepared by RAFT polymerization, is a facile and effective means to improve the dispersion stability of the SPIO and CNT hybrid materials particularly in PBS. The CNT hybrid materials exhibit low in vitro cytotoxicity, with up to a concentration of 200 µg mL1

, and low in vivo acute toxicity in mice with doses up to 100 mg kg-1. The hybrid material CNT-

Fe3O4-P4 exhibits a superparamagnetic behaviour with a saturation magnetization about 11 emu g-1 at room temperature, attributing to the TB temperature of 200 K. Although the R2 relaxivity decreases with the increase of DP of diblock copolymer, the CNT-Fe3O4-P4 shows a greater contrast enhancement in vivo with about 55% increase in tumor/liver contrast ratio after administration. Thus, the modified MWCNTs are promising candidates as a scaffold to load magnetic nanoparticles for applications as MRI contrast agents. The in vivo animal trials show that the CNT-Fe3O4-P4 hybrid material is of great potential for use as an MRI contrast agent, featuring improved PBS dispersibility, increased tumor/liver contrast ratio, non-cytotoxicity, and good in vivo tolerance.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]; [email protected] ACKNOWLEDGMENT


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This work was financially supported by Australia-China Special Fund (CH090192) and the NSFC (Nos. U1203292, 21361162004). YL thanks the China Scholarship Council (CSC) and CSIRO for providing scholarships. BAM was supported by the Australian NHMRC (56992, 50932). The authors wish to acknowledge Miss Fengxiang Qie (CSIRO and Beijing University of Chemical Technology), Miss Xuan Nguyen (CMSE, Clayton), and Mr Marcio Pasotto (CMSE, Clayton) for their help. The authors also acknowledge Barry Halstead for XRD, Yesim Gozukara for ICPMS, Keith Murray and Boujemaa Moubarak for SQUID measurements, and Molecular Imaging In. for animal work and in vivo MRI imaging. The authors thank Dr Jackie Cai for helpful discussions about the manuscript.

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Table of Contents Graphic


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Colloidally Stabilized Magnetic Carbon Nanotubes ... - ACS Publications

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